View Abstract

A2666 - Enhancing Regenerated Tissue Maturation and Graft Function Through Ex Vivo Biomimetic Culture in Automated Multi-Channel Rat Lung Bioreactor
Author Block: D. Gorman, T. Wu, H. C. Ott, S. E. Gilpin; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA, United States.
Rationale: Recellularization of acellular native lung scaffolds is a promising approach for lung tissue bioengineering. Currently, appropriate cells are delivered to the scaffold airways and vascular network, but without further direction of cell location or distribution. Following seeding, lungs are cultured ex vivo with vascular perfusion of media to maintain cell viability. During lung culture, the addition of mechanical cues may provide additional regenerative benefit, by driving cellular proliferation and organization within the developing tissue. We examined techniques to deliver additional biomechanical and biophysical stimuli during ex vivo regeneration, studying the impact of both fluid and air ventilation during culture. We hypothesized that both fluid and air ventilation would aid cell viability and lung tissue formation, when compared to vascular-perfused lungs in the absence of ventilation.
Methods: In parallel, rat lung scaffolds seeded with primary human pulmonary artery endothelial cells and basal epithelial cells were cultured for up to one week using a multi-channel small animal bioreactor system. All lungs were cultured 2 days in the absence of ventilation followed by a 4-day period with differing ventilation techniques which included fluid ventilation, air ventilation, air ventilation with supplemental porcine surfactant, and no ventilation as a control. Tissue regeneration was analyzed by resazurin metabolic assay, histology, and qPCR.
Results: Resazurin metabolic assay revealed a trend toward increased cell number during 4 days of no ventilation (+1.23±0.15-fold) while early air ventilation (-3.75±3.59-fold), air ventilation with surfactant (-14.88±2.09-fold), and fluid ventilation (-40.11±40.02-fold) showed a decrease in cell number. The addition of surfactant to air ventilated lungs aided in lung recruitment and reduced airway pressure during ventilation.
Despite lower total cell numbers, qPCR analysis revealed increased Ki67 gene expression in air ventilated (+6.82±1.31-fold) and fluid ventilated (+2.70±1.02-fold) lungs as compared to the perfusion-only control lungs. An increase in expression of CYR61, a gene downstream of the mechanosensing YAP signaling pathway, was also upregulated in in air ventilated (+2.70±0.61-fold) and fluid ventilated (+1.76±0.33-fold) lungs as compared to the control lung. This suggests a cellular response to varied ECM mechanics during cyclic ventilation, which can be harnessed to further control cell spreading, migration, and proliferation during ex vivo lung culture.
Conclusions: We conclude that an initial vascular perfusion-only culture period, without airway ventilation, is important for lung epithelial regeneration, but that a multi-phase culture protocol that subsequently introduces air ventilation can be used to drive more prolonged cell proliferation and functional lung tissue organization.